Dynamic Analysis of Cortisol Hormone, Alpha-Amylase Enzyme, and Blood Lactate Levels during a Rowing Ergometer 6 km Race

Abstract

Rowing races require extreme physical and psychological effort from every athlete. This study aimed to determine the dynamics of the salivary cortisol and alpha-amylase, as well as blood lactate throughout the specific load represented by a 6 km rowing race, conducted on a rowing ergometer. The sample consisted of 11 junior and senior rowers from HVK Gusar in Split (n = 11) who actively competed at club and international levels. Variables consisted of three repeated oral samples of the hormone cortisol and the enzyme alpha-amylase determined in saliva and three repeated blood lactate samples. Potential differences in the levels of the studied variables at different time points were determined using a repeated-measures ANOVA test. The results showed different dynamics of hormonal (cortisol) and metabolic (alpha-amylase and lactates) variables. All variables experienced a significant post-race increase, while other changes were not significant. The results highlighted that high-intensity rowing causes an increase in the body’s cortisol, alpha-amylase, and lactate levels. This should be implemented in rowing training to find the right balance between high and low-intensity rowing, enabling athletes’ progression while reducing the risk of overtraining.

1. Introduction

Rowing is an exceptionally demanding Olympic sport with a standardized race length of 2000 m, equaling around 250 strokes, and lasting from 6 to 7 min on average. Each stroke must be perfectly executed with minimal variation in technique and power [1]. Typically, the race requires the involvement of both aerobic and anaerobic energy pathways and high levels of physical and mental strength to resist pain, delay exhaustion, and eventually win the race [2]. It has been shown that around 77% of total energy demand depends on aerobic energy pathways, while the remaining 23% is anaerobic, with muscle glycogen degradation playing a crucial role [3]. Due to the mentioned sports specificities, rowers are usually tall and muscular, with extremely developed cardiorespiratory systems, a high percentage of slow twitch muscle fibers, and a low body fat percentage [4]. Prolonged high-intensity rowing induces various physiological reactions. It triggers the body’s cardiorespiratory, endocrine, and metabolic responses to maintain the effort and resist failure. This results in the progressive involvement of anaerobic energy pathways, followed by eventual lactate accumulation [5]. Alongside the activation of the cardiovascular and respiratory systems, a vast array of hormones is released. Measuring certain biomarkers such as lactate from the blood or hormone cortisol and enzyme alpha-amylase from saliva might help us optimize training for the athletes. It is accepted that measuring hormonal response during activities should be a valid tool for monitoring the demands, loads, and stresses that athletes endure [6,7].

Blood is a major component of the cardiovascular system. The cell portion of the blood is called hematocrit, consisting of red blood cells, white blood cells, and platelets, taking approximately 45% of the blood’s total volume [8]. The oxygen-carrying capacity of red blood cells depends on hemoglobin, a specialized protein that binds and transports oxygen and carbon dioxide [9]. Higher training intensities provoke the development of energy from anaerobic sources, increasing heart rate and greater blood circulation, as well as lactate accumulation. Lactate is a metabolic by-product of anaerobic energy development [10]. During glycolysis, blood glucose or muscle glycogen is converted to pyruvate, which enters the mitochondria to continue aerobic respiration or get converted to lactate, depending on the exercise’s intensity and oxygen availability [11]. As the intensity of the exercise increases and oxygen availability reduces, energy development becomes more anaerobic and is followed by higher lactate production [5]. Measuring an athlete’s lactate can give us useful information about individual effort and metabolic status. Held et al. [12] showed that the maximal lactate accumulation rate is linked to lactic anaerobic performance and rowing power production.

The endocrine responsiveness of athletes is directly related to physical exertion, which can induce both physical and psychological stress, depending on various conditions [13]. Since the hormone cortisol and enzyme alpha-amylase respond to both stress forms, simultaneous measurements can help to evaluate sport-specific psycho-physiological demands. Both cortisol and alpha-amylase are convenient markers for stress research, indicating the body’s sympathetic activity. Both markers could be non-invasively measured in saliva allowing sampling in various settings. Cortisol, commonly known as the stress hormone reflects the activation of the hypothalamic–pituitary–adrenocortical axis, which is directly related to physiological stress response, providing catabolic effects [14,15]. Because of that, cortisol is a frequently used biomarker of both physical- and mental-stress-related functioning of athletes [16]. Recent research reported a blunting of the cortisol response in elite rowers during competition, which may indicate maladaptive stress responses [17]. It was also found that testosterone and cortisol are more sensitive to changes in training volume than growth hormone and perceived recovery stress state [18]. Alpha-amylase enzyme, found in saliva, aids in the breakdown of complex carbohydrates into simpler molecules such as maltose. Exercise can influence the releasing rate of an alpha-amylase enzyme [19], and it has been shown that this effect is more obvious at exercise intensities higher than 70% VO₂ max [20]. Also, alpha-amylase can reflect the activity of the sympathetic nervous system because it is highly related to increased levels of noradrenalin and eventually reflects the state of arousal [21]. It has been found that cortisol and alpha-amylase levels were suppressed in ultra-marathon runners as a possible response to prolonged physical stress [22].

To the best of our knowledge, only one study involving rowing simultaneously examined the dynamics of alpha-amylase and cortisol in competitive conditions, and the results indicated a significant increase in both biomarkers during the 2 km ergometer rowing race [19]. It was also found that athletes with the lowest levels of α-amylase and cortisol reactivity to the competition reported the highest perceived dominance. In contrast, those with low α-amylase and high cortisol reactivity reported the lowest perceived dominance [19]. Most of the scientific rowing research includes the rowing ergometer, a machine that simulates rowing on-water movement with high accuracy while producing instant feedback about every stroke, allowing greater control of the training process. Most existing studies focused on the 2 km rowing ergometer race, while this study’s focus was on the 6 km race, with a greater aerobic component, mostly used during the early season to evaluate specific aerobic capacities of the athletes [23,24]. Because of that, this study aimed to evaluate the endocrine (cortisol) and metabolic (alpha-amylase and blood lactate) responsiveness to specific rowing indoor competition stress of a 6 km ergometer race and tries to elucidate possible associations of levels among included biomarkers.

2. Materials and Methods

2.1. Participants

This study included 11 (junior and senior) male rowers from HVK Gusar Split (age 17.2 ± 2.31 years on average). All participants were competing on club and national level, training regularly for at least 12 h per week. The weekly training program consisted mostly of rowing sessions, both outdoor and indoor, with additional resistance training performed in the gym to reach desired neuromuscular adaptations. A test consisted of 6 km of rowing performed on a rowing ergometer. The study was conducted during the early season as a part of regular rowing physical-testing procedures. This generated high motivation levels among rowers since their performance was not only important for the study’s purposes but also as a team selection for the following season. Participation in this study was voluntary. All rowers were informed about the purpose of the study and signed a consent form before the study. The study was approved by the ethical board of the University of Split, Faculty of Kinesiology (Approval No. 2181205-02-05-23-002).

2.2. Procedures and Variables

Apart from anthropometrics (height, body mass, and body composition), variables in this study included 6 km test results in terms of average 500 m speed, heart rate measures, and blood (lactate) and salivary (cortisol and alpha-amylase) biomarkers. All participants performed the test on Concept 2 rowing ergometers, with the drag factor set to 120. Salivary biomarkers of hormonal status are thought to be non-invasive in providing an objective, rapid, and reliable evaluation of the endocrine system in athletes [25,26]. In this research, salivary and blood samples were taken before the testing, after the warm-up procedure, and after the testing. All participants were familiar with the study requirements, and they were introduced to certain guidelines regarding their behavior before the study. The warm-up procedure was standardized. It involved 20 min of aerobic, steady-state rowing with a tempo not higher than 20 (strokes per minute), including 3 higher intensity intervals (corresponding to the 12th, 14th, and 16th minute of 20 min warm-up) limited to only 10 race-pace strokes. Rowers had rested 48 h after the previous training session. After fasting overnight and avoiding a substantial meal for 60 min before sample collection, rowers thoroughly rinsed their mouths with water 10 min before each sample was taken. During this time, athletes were not permitted to consume any fluids to ensure a standardized sampling procedure. After the samples were collected, participants were allowed to drink freely. The SalivaBio Oral Swabs (Salimetrics LLC, State College, PA, USA) were used, placed under the tongue on the floor of the mouth for 2 min. Once collected, swabs were put into storage tubes and immediately refrigerated. Within 2 h, samples were frozen at temperatures below −20 °C until the day of analysis. On the analysis day, samples were fully thawed and centrifuged at 1500× g (3000 rpm) for 15 min. Following centrifugation, assays were conducted. Saliva cortisol was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) from Salimetrics LLC (State College, PA, USA) with a micro-plate reader (Infinite 200PRO, Tecan, Männedorf, Switzerland). Standard curves were developed according to the manufacturer’s instructions, using commercially available standards and quality-control samples for all assays. Alpha-amylase levels were analyzed with a kinetic enzyme assay kit (Salimetrics LLC, State College, PA, USA). The measured concentrations were adjusted for the salivary flow rate to reduce the impact of dehydration. Capillary blood samples were taken from the fingertip at three time points (baseline, post-warm-up, and post-race) and analyzed using a lactate analyzer (Biosen C-Line; EKF Diagnostics GmbH, Barleben, Germany).

2.3. Statistical Analysis

The normality of the data was tested using the Kolmogorov–Smirnov test procedure. Homoscedasticity was checked by the Levene test. The differences among measurements of blood and salivary biomarkers were calculated by analysis of variance for repeated measurements (ANOVA) with subsequent Bonferroni post hoc test analyses. All the analyses were performed using Statistica v.14.0.1.25 (TIBCO Software Inc., Greenwood Village, CO, USA), and the significance level was set to p < 0.05.

3. Results

The results of descriptive statistics showed the central tendencies and standard deviations of the studied variables, which are visible in the attached

Table 1. Descriptive statistics.

Variable	Mean	SD	Min	Max	Median	K-S (p)
BH (cm)	185.10	3.07	182	196	185	p > 0.200
BM (kg)	85.65	9.10	71.4	103.7	84.5	p > 0.200
BF%	17.47	3.92	8.8	22.9	17.2	p > 0.200
MM (kg)	39.33	2.24	36.7	43	39.3	p > 0.200
MM%	46.16	2.77	41.5	51.6	46	p > 0.200
6 km (s)	1302.94	44.60	1210.3	1354.3	1317.1	p > 0.200
LA 1 (mmol/L)	1.48	0.29	1	2	1.5	p > 0.200
LA 2 (mmol/L)	2.10	0.60	1.3	2.9	1.9	p > 0.200
LA 3 (mmol/L)	11.81	2.38	7.5	14.9	12.1	p > 0.200
AA 1 (U/mL)	271.89	206.29	26.8	566.4	183.6	p > 0.200
AA 2 (U/mL)	233.54	136.42	35.3	433.8	168.9	p > 0.200
AA 3 (U/mL)	581.99	395.28	86.2	1137.3	487.8	p > 0.200
COR 1 (μg/mL)	0.68	0.25	0.3	1.1	0.7	p > 0.200
COR 2 (μg/mL)	0.51	0.24	0.2	0.9	0.5	p > 0.200
COR 3 (μg/mL)	0.94	0.41	0.4	1.7	0.8	p > 0.200
HR BAS (beats/min)	115.00	12.81	98	136	114	p > 0.200
HR AVE (beats/min)	186.42	9.05	170	203.2	188	p > 0.200
HR MAX (beats/min)	201.33	6.76	192	213	200	p > 0.200

Legend: SD—standard deviation; Min—minimum; Max—maximum; K-S (p)—p-value of K-S normality test; BH = body height; BM = body mass; BF% = body fat percentage; MM = muscle mass; MM% = muscle mass percentage; 6 km = 6 km test result; LA 1 = initial lactate sample; LA 2 = second lactate sample; LA 3 = third lactate sample; AA 1 = initial alpha-amylase sample; AA 2 = second alpha-amylase sample; AA 3 = third alpha-amylase sample; COR 1 = initial cortisol sample; COR 2 = second cortisol sample; COR 3 = third cortisol sample; HR BAS = initial heart rate; HR AVE = average heart rate; HR MAX = maximum heart rate.

. Using the Kolmogorov–Smirnov test, it was determined that all variables are normally distributed.

Table 2. ANOVA for repeated measures.

	F	p	(ηp2)
AA	10.92	0.001	0.55
COR	12.58	0.001	0.58
LA	195.25	0.001	0.96

Legend: AA—alpha-amylase; COR—cortisol; LA—lactates; F—f value; p—p-value; ηp²—effect size value.

shows the ANOVA results for biochemical parameters. A nonsignificant decrease in cortisol levels is seen on Figure 1, which was reported after the warm-up protocol, followed by a significant increase (p = 0.001, ηp² = 0.58) after the 6 km rowing race.

Alpha-amylase values, as presented in Figure 2, experienced a nonsignificant decrease after the warm-up protocol, followed by a significant increase (p = 0.001, ηp² = 0.55) after the 6 km rowing race.

The levels of measured blood lactate (Figure 3) showed a nonsignificant increase after the warm-up protocol, followed by a significant increase (p = 0.001, ηp² = 0.96) after the 6 km rowing race.

4. Discussion

The results highlighted the different dynamic responses of the included variables to the testing procedure. The dynamic responses of cortisol hormone and alpha-amylase enzyme were similar, dropping from baseline levels to post-warm-up levels, followed by post-race levels’ increase. On the other hand, blood lactate levels progressed gradually with every sampling, rising from baseline levels to post-warm-up levels, followed by another increase detected after the end of the race. Considering the different physiological backgrounds among the variables, it could be concluded that the 6 km rowing ergometer race elicited different hormonal and metabolic responses.

4.1. Stress Biomarkers

Because the standardized warm-up protocol included 20 min of steady-state low-intensity rowing, the decline in cortisol and alpha-amylase levels after the warm-up may be explained by the effects of the low-intensity aerobic stimulus. Hill et al. [27] showed that cortisol response differentiates between exercise intensities, rising with high- and moderate-intensity exercise (corresponding to 60% VO₂ max and higher) and decreasing with low-intensity exercise (equivalent to 40% VO₂ max). Moreover, the rowing sport is characterized by rhythmic cyclic movements to maintain a specific number of strokes per minute, and it was found that rhythmic aerobic exercise can cause a decrease in alpha-amylase levels over time [28]. Since higher alpha-amylase levels indicate sympathetic nervous system activation and parasympathetic inhibition, it can be speculated that the rhythmic steady-state warm-up activity resulted in increased parasympathetic activity, reduced stress in the athletes, and decreased alpha-amylase levels [29]. Post-race hormonal response aligns with this explanation as cortisol and alpha-amylase levels were influenced by prolonged high-intensity rowing, affected by strong sympathetic activation [29]. Previous studies showed that low-intensity exercise does not influence cortisol levels in moderately trained men, while moderate-to-high-intensity efforts result in a cortisol increase [27]. In 2014, Koibuchi and Suzuki [20] found that the intensification of carbohydrate metabolism triggered by high energy demands results in a higher rate of alpha-amylase release. Rowing a 6 km race represents a great submaximal physical effort, which is why both variables experienced a significant post-race increase. This finding seems to follow Kallen et al. [30], who also reported higher post-race cortisol levels in competitive rowers compared to baseline levels. Their study included 16 competitive lightweight (n = 8) and heavyweight (n = 8) rowers who performed the 2000 m on-water rowing race. Although our study involved an ergometer indoor race over 6000 m with a greater aerobic component and duration, the results of both studies suggest that cortisol levels increase after high-intensity rowing stimulus. Similar cortisol dynamics are reported in other sports with different physical demands, with an increase in cortisol levels from pre- to post-match rise in football (85%), basketball (61%), and rugby (71%) [6,31,32]. Since both cortisol hormone and alpha-amylase enzyme indicate sympathetic activity, they reported similar competitive environment dynamics.

4.2. Blood Lactate Levels

The metabolic response in terms of lactate accumulation increased gradually throughout the testing procedure. Lactate production is a result of the progressive demand for energy development from anaerobic sources, which was minimal at the baseline point, slightly higher during the warm-up procedure, and significantly higher during the race [5,10]. The first increase in lactate levels is a result of a warm-up procedure, although previous research showed that performing low-intensity exercise after higher-intensity anaerobic efforts can help reduce blood lactate levels [33]. Despite a low steady-state intensity, the warm-up period initiated greater muscle activation and slight anaerobic energy development from some muscle groups, leading to a slight lactate increase. Since athletes followed specific preparation guidelines and arrived at the club well-rested, baseline lactate levels were not high enough for them to be decreased by a low-intensity warm-up period. Also, the warm-up protocol involved a few short high-intensity intervals consisting of not more than 10 strokes, which could have been the reason for a slight lactate level increase. The post-race lactate spike could be explained by the higher rowing intensity. Lactate production is a result of progressive demand for energy development from anaerobic sources, and it has been shown that anaerobic glycolytic pathways gain importance at the beginning and the end of the race, with aerobic glycolysis dominating in between [2]. During a 6 km rowing ergometer race, athletes sustained high efforts for a prolonged time, topped by a final sprint to reach the best results possible. This influenced greater lactate accumulation, leading to a more significant post-race lactate level. These findings follow a 2009 study from de Campos Mello et al. [34], who found that there is no significant difference in peak lactate levels between rowing racing performed on water or an ergometer, with average post-race lactate accumulation higher than 10 mmol L-1 over the 2000 m distance. Although different race lengths were included, both studies recorded significant post-race lactate increases caused by the anaerobic energy demands at the beginning and the end of the race [2].

4.3. Strengths and Limitations

The limitations of this study include the absence of data on athletes’ anabolic response, as salivary testosterone was not measured, unlike cortisol and alpha-amylase. Additionally, there is a lack of data regarding athletes’ psychological responses, such as anxiety or self-confidence before the race, which could potentially influence biomarker levels. Furthermore, baseline values for the studied biomarkers on non-race days were not included, despite baseline salivary and blood biomarker values being measured upon arrival. Finally, the greatest limitation of the study could be seen through the small sample consisting of only 11 rowers, due to the lack of Croatian junior and senior club rowers in general.

The professional athlete profiles, simultaneous evaluation of three biomarkers, and measurements performed at three time points (baseline, post-warm-up, and post-race) are important strengths of this research. Moreover, the study was integrated into regular early season testing procedures, which kept athletes highly motivated, since the quality of their performance determined their chances of making the top crews in the upcoming season. The main strength comes from the fact that most scientific work is focused on a 2 km race distance, with not many studies including simultaneous measurement of salivary cortisol and alpha-amylase. Our study examined the mentioned biomarkers during a 6 km race with a greater aerobic component, allowing us to draw conclusions about hormonal and metabolic responses to specific demands of this rowing test mostly used during the early season.

Future lines of research should aim to include certain psychological and anabolic variables to complement the current findings of the situational stress of a 6 km ergometer race, as well as include the wider sample and female rowers.

5. Conclusions

The testing procedure was composed of a specific warm-up protocol followed by a 6 km rowing ergometer race. This test triggered different hormonal and metabolic responses in athletes. Samples of saliva cortisol and alpha-amylase, as well as blood lactate, were collected at three time points (baseline, post-warmup, and post-race). Low-intensity warm-up protocol influenced a slight decrease in cortisol and alpha-amylase levels with only slight increases in blood lactate levels. Rowing a 6 km ergometer race triggered the athletes’ metabolic and hormonal responses and influenced significant post-race increases in all measured variables.

Monitoring of metabolic and hormonal responses may be used to determine individual efforts and stress that athletes endure, optimizing training load and creating adequate psychological interventions. This can help coaches control the overreaching process, avoiding negative overtraining effects. Moreover, the results suggest that low-intensity rowing could be a potential tool for acute psychological stress decrease.

In future studies, it would be beneficial to analyze testosterone levels to assess the anabolic component of hormonal response, as well as to include certain psychological tests for a better understanding of an individual stress response.